Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line.
Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBankTM data base (human HNP36, 326 residues) that has been identified as a growth factor-induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5'-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter.
Crawford
C R
CR
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Patel
D H
DH
Naeve
C
C
Belt
J A
JA
D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D033722:Equilibrative-Nucleoside Transporter 2; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D009705:Nucleosides / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014529:Uridine / Q000378:metabolism
D000345:Affinity Labels; D002352:Carrier Proteins; D033722:Equilibrative-Nucleoside Transporter 2; D008565:Membrane Proteins; D009687:Nuclear Proteins; D009705:Nucleosides; C438654:SLC29A2 protein, human; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D014529:Uridine